Mapping chromatin structure in vivo using DNA methyltransferases

Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2004-05, Vol.33 (1), p.68-80
Main Authors: Jessen, Walter J, Dhasarathy, Archana, Hoose, Scott A, Carvin, Christopher D, Risinger, April L, Kladde, Michael P
Format: Article
Language:English
Subjects:
Acid Phosphatase
Chromatin - chemistry
Chromatin - genetics
Chromatin - metabolism
Deamination
DNA (Cytosine-5-)-Methyltransferases - metabolism
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Molecular Probes
Polymerase Chain Reaction
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - metabolism
Sequence Analysis, DNA - methods
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Summary:Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary uses for the DNA methyltransferase CviPI (M.CviPI, GC specificity) in probing chromatin organization. First, we describe the use of the naturally occurring, free enzyme as a diffusible probe to map changes in nucleosome structure and to footprint factor interactions at cis-regulatory sequences. In a second application, termed targeted gene methylation (TAGM), the DMTase is targeted via in-frame fusion to a DNA-binding factor. The rapid accumulation of DNA methylation enables highly sensitive detection of factor binding. Both strategies can be applied with any C5 DMTase, such as M.SssI, which also possesses a short-recognition specificity (CG). A description of methods for constructing C5 DMTase-expressing strains of Saccharomyces cerevisiae and analyzing chromatin regions is provided. We also include comprehensive protocols for the isolation and bisulfite treatment of genomic DNA as well as the subsequent bisulfite sequencing steps. Data demonstrating the efficacy of both DMTase probing techniques, theoretical considerations, and experimental analyses are presented at GAL1 and PHO5.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2003.10.025